Cytotoxic T-lymphocytes (CTL) reactive against human melanoma recognize a set of epitopes that consist of peptides associated with class I MHC molecules. These peptide epitopes are derived from a number of cellular proteins, including the shared melanocytic differentiation antigens (MDAs) tyrosinase, gp100/Pmel17, MART-1/Melan-A, and gp75/trp-1. Additional shared peptide epitopes and an unknown number of unique epitopes that are HLA-A2-associated remain to be identified. It is not clear that the most immunodominant peptides have all been identified. The purpose of the proposed work is to expand what is known about peptide epitopes for melanoma-reactive CTL, to assess the clinical importance of immune responses to MDA-peptides, and to evaluate peptide-based tumor vaccines in patients with high risk melanoma. The Specific Aims are as follows: Aim 1: To identify peptide epitopes for melanoma-reactive CTL. This will include (a) direct identification of epitopes for melanoma-reactive human CTL restricted by HLA-A2 or by HLA-A3, using a combination of cellular methods and tandem mass spectrometry, and (b) identification of CTL reactivity against epitopes derived from defined melanoma lineage proteins. Aim 2: To determine the clinical significance of CTL responses to known HLA-A2-restricted peptide epitopes for melanoma reactive CTL in patients with melanoma (a) to determine whether p946 and other peptides derived from melanocytic tissue differentiation antigens are recognized on normal melanocytes by melanoma-reactive human CTL; (b) to determine whether CTL responses to these peptides in melanoma patients correlates with stage of disease and prognosis. Aim 3: To determine whether human CTL responses to a defined melanoma peptide can be augmented by vaccination in the adjuvant setting; (a) to determine whether CTL reactivity to peptide 946 can be enhanced by vaccination with peptide-based vaccines, (b) to determine whether vaccination with a vaccinia construct offers greater immunogenicity than an optimized purified peptide vaccine.